life Article Inferring the Phylogenetic Positions of Two Fig Wasp Subfamilies of Epichrysomallinae and Sycophaginae Using Transcriptomes and Mitochondrial Data Dan Zhao , Zhaozhe Xin, Hongxia Hou, Yi Zhou , Jianxia Wang, Jinhua Xiao * and Dawei Huang * Institute of Entomology, College of Life Sciences, Nankai University, Tianjin 300071, China; [email protected] (D.Z.); [email protected] (Z.X.); [email protected] (H.H.); [email protected] (Y.Z.); [email protected] (J.W.) * Correspondence: [email protected] (J.X.); [email protected] (D.H.); Tel.: +86-185-2245-2108 (J.X.); +86-139-1025-6670 (D.H.) Abstract: Fig wasps are a group of insects (Hymenoptera: Chalcidoidea) that live in the compact syconia of fig trees (Moraceae: Ficus). Accurate classification and phylogenetic results are very important for studies of fig wasps, but the taxonomic statuses of some fig wasps, especially the non-pollinating subfamilies are difficult to determine, such as Epichrysomallinae and Sycophaginae. To resolve the taxonomic statuses of Epichrysomallinae and Sycophaginae, we obtained transcrip- tomes and mitochondrial genome (mitogenome) data for four species of fig wasps. These newly added data were combined with the data of 13 wasps (data on 11 fig wasp species were from our laboratory and two wasp species were download from NCBI). Based on the transcriptome and genome data, we obtained 145 single-copy orthologous (SCO) genes in 17 wasp species, and based on mitogenome data, we obtained 13 mitochondrial protein-coding genes (PCGs) for each of the 17 wasp species. Ultimately, we used 145 SCO genes, 13 mitochondrial PCGs and combined SCO genes and mitochondrial genes data to reconstruct the phylogenies of fig wasps using both maximum likelihood Citation: Zhao, D.; Xin, Z.; Hou, H.; Zhou, Y.; Wang, J.; Xiao, J.; Huang, D. (ML) and Bayesian inference (BI) analyses. Our results suggest that both Epichrysomallinae and Inferring the Phylogenetic Positions Sycophaginae are more closely related to Agaonidae with a high statistical support. of Two Fig Wasp Subfamilies of Epichrysomallinae and Sycophaginae Keywords: fig wasps; classification; phylogeny; mitochondrial gene; transcriptome Using Transcriptomes and Mitochondrial Data. Life 2021, 11, 40. https://doi.org/10.3390/life11010040 1. Introduction Received: 18 December 2020 Fig wasps (Hymenoptera: Chalcidoidea) refer to all wasps that must rely on the Accepted: 9 January 2021 syconia of fig trees (Moraceae: Ficus) to complete their life histories. According to whether Published: 11 January 2021 they pollinate the figs, fig wasps are broadly classified into two categories of pollinating fig wasps and non-pollinating fig wasps [1]. Among them, the symbiosis of figs–pollinating Publisher’s Note: MDPI stays neu- fig wasps is a classical model of the mutualistic system, which originated about 75 million tral with regard to jurisdictional clai- years ago [2]. The plant–insect interaction system between figs and fig wasps provides ms in published maps and institutio- nal affiliations. an ideal model for the study of the co-evolution of species and symbiotic relationship among organisms [3], and these studies are inseparable from the correct identification and phylogenetic history reconstruction of fig wasps. The current taxonomic information listed on the fig web (http://www.figweb.org/ Copyright: © 2021 by the authors. Li- Fig_wasps/Classification/index.htm) is mainly based on the taxonomy system proposed censee MDPI, Basel, Switzerland. by Heraty [4], in which the fig wasps are classified into five families (Agaonidae, Eurytomi- This article is an open access article dae, Ormyridae, Pteromalidae, Torymidae) and ten subfamilies (Agaoninae, Kradibiinae, distributed under the terms and con- Sycophaginae, Tetrapusiinae, Colotrechinae, Pteromalinae, Epichrysomallinae, Otitesel- ditions of the Creative Commons At- linae, Sycoecinae, and Sycoryctinae) [4]. The taxonomic statuses of some subfamilies tribution (CC BY) license (https:// are difficult to determine, such as the non-pollinating wasps of Epichrysomallinae and creativecommons.org/licenses/by/ Sycophaginae [5–8]. 4.0/). Life 2021, 11, 40. https://doi.org/10.3390/life11010040 https://www.mdpi.com/journal/life Life 2021, 11, 40 2 of 11 The taxonomic history of Epichrysomallinae and Sycophaginae has undergone multi- ple changes since the beginning of the taxonomy of fig wasps in the 19th century. Sycophag- inae was founded by Walker in 1875 [9], then it was included in the family of Torymidae based on cleptoparasitic habits by Joseph in 1964 [10]. Epichrysomallinae was established and classified in Torymidae by Hill in 1967, who also agreed to classify Sycophaginae in To- rymidae [5]. In 1981, Bouˇcekrevised and adjusted Epichrysomallinae into Pteromalidae on the basis of morphological characteristics [6]. In 1988, Bouˇcekfurther considered the whole Agaonidae as a monophyletic group based on the reproductive characteristics, of which the Agaoninae was only the most specific group with the behaviors of pollination, and thus both Epichrysomallinae and Sycophaginae were classified in Agaonidae [7]. In 1998, Rasplus et al. used the D1 and D2 domains of nuclear 28S rRNA to infer the phylogenetic relationships of six subfamilies proposed by Bouˇcek,and they indicated that Agaonidae that Bouˇcek(1988) referred to was not a monophyletic group [8]; they further revised Agaonidae and pointed out that Agaonidae only contained the subfamily of Agaoninae, and the taxonomic statuses of Epichrysomallinae and Sycophaginae in Chalcidoidea were undetermined. In 2013, Heraty et al. used 233 morphological and two molecular datasets (nuclear ribosomal 18S and 28S D2-D5 expansion regions) to make phylogenetic inference of a variety of wasps in Chalcidoidea and the results showed that the two subfamilies of Epichrysomallinae and Sycophaginae previously undetermined were included in Ptero- malidae and Agaonidae, respectively [4]. In 2018, Peters et al. constructed a phylogenetic tree using 3239 homologous genes based on transcriptomic data from 62 species of Chalci- doidea, which showed that Epichrysomallinae was more closely related to Agaonidae than Pteromalidae, but Sycophaginae was not involved in their studies [11]. Therefore, due to dif- ferent sources of taxonomic evidences (morphology, biological characteristics or molecular evidences), the taxonomy and phylogenetic positions of the two subfamilies of Epichryso- mallinae and Sycophaginae, especially the latter, are still unclear, and more data are needed to clarify whether they belong to Agaonidae or Pteromalidae. The second-generation high-throughput sequencing technology represented by tran- scriptome RNA sequencing (RNA-seq) can obtain a large-scale sequencing of the transcripts of specific tissues of a certain species. By using bioinformatics tools for splicing assem- bly, we can quickly obtain almost all the gene coding sequences (CDS) of the specific tissue of the species at a time point. With the advantages of low cost, large data volume, high efficiency and high accuracy, RNA-seq has shown great potential in the field of molec- ular phylogenetic research and become an effective means for molecular biology research of non-model animals [12–14]. The mitochondrial genome (mitogenome) sequences are also widely used in molecular evolution, phylogeny, phylogeography and population genetics because of their advantages, such as small genome size, maternal inheritance, no intron, relatively high evolutionary rate, simple structure, conserved gene content, and rare recombination [15–17]. In this study, we obtained conserved single-copy orthologous (SCO) genes in the nucleus based on transcriptomes (Odontofroggatia galili, Walkerella microcarpae, Micranisa ralianga, Platyneura mayri and Encarisa Formosa) and genomes (another 12 wasp species) from 17 wasp species and mitogenome sequences based on second-generation genome sequencing. Phylogenies of fig wasps were reconstructed based on three different molecular datasets, conserved SCO genes in the nucleus, 13 mitochondrial protein-coding genes (PCGs) and combined genes (SCO genes combined with 13 mitochondrial PCGs) to obtain new evidences to explore the taxonomic status of Epichrysomallinae and Sycophaginae. 2. Materials and Methods 2.1. Taxon Sampling and Data Collection A total of 17 wasp species were included in this study (Table1), including 15 fig wasp species (representing three families and six subfamilies). Four fig wasp species (O. galili, W. microcarpae, M. ralianga and P. mayri) were newly sequenced in this study. Data on 11 fig wasp species were from our laboratory and 2 wasp species were download from Life 2021, 11, 40 3 of 11 NCBI. Among the four species, O. galili, W. microcarpae, and M. ralianga were collected from Qinzhou, Guangxi, China (N21◦570, E108◦370), and P. mayri was collected from Xishuangbanna, Yunnan, China (N21◦410, E101◦250). The information on the host fig trees was as follows: O. galili and W. microcarpae were associated to Ficus microcarpa, P. mayri was associated to Ficus racemosa, and M. ralianga was associated to Ficus altissima. We collected the figs in their natural state of maturity (but fig wasps have not left the fig yet) in the wild. All the fig wasps were collected after they came out from the figs, and then identified by using the SMZ-168 microscope (Motic, China). The identification of fig wasps was based mainly on descriptions and pictures in the literatures [7,18], and also combined with photographs of fig